Niemann-Pick Type C (NPC) disease is a rare, pediatric, genetically recessive neurological disease. NPC is caused by mutations in genes known as either Npc1, which encodes a membrane protein with 13-16 transmembrane domains (TMD), or, in approximately 5% of patients, Npc2, which encodes a soluble protein. Loss of function in either the NPC1 or NPC2 protein results in clinically indistinguishable disease phenotypes, with accumulation of cholesterol, sphingomyelin, sphingosine, and gangliosides GM2 and GM3 within the late endosomes/lysosomes. This accumulation of lipids results in progressive neurodegeneration, hepatomegaly and splenomegaly, and ultimately early death, usually before age 20 years. Currently, this disease has no cure.
NPC1 and NPC2 are endosomal cholesterol binding proteins that work in concert to transport cholesterol out of the late endosomes/lysosomes to various cellular compartments, including the endoplasmic reticulum (ER). Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a resident enzyme located at the ER that utilizes cholesterol and fatty acyl-coenzyme A as its substrates to produce cholesteryl esters. ACAT1 is responsible for the bulk of cellular cholesteryl esters found in various cells. Lacking functional NPC1 or NPC2 considerably slows the transport rate of cholesterol from the late endosomes/lysosomes to the ER. However, through NPC1/NPC2-independent mechanisms for delivering cholesterol, enough cholesterol eventually arrives at the ER for esterification. Thus, a significant amount of cholesteryl ester is still present in mutant NPC cells.
Several mouse models for NPC disease are available. In the original NPC1−/− mice, alteration within the Npc1 gene causes a frame-shift mutation and results in premature truncation of the NPC1 protein (Loftus et al. 1997. Science 277:232-235). Neuronal cholesterol accumulation occurs as early as postnatal day (PND) 9 in the mouse model (Reid et al. 2004. J. Lipid Res. 45:582-591), then loss of Purkinje neurons in the cerebellum begins to occur shortly after weaning (at PND 21) (German et al. 2001. Neuroscience 105:999-1005). The mice begin to lose weight at 7 weeks of age, and begin to die at 11 weeks of age. In a newly discovered mutant NPC mouse model (the npc1NMF164 mouse, also referred to as the Npc1m/m mouse), a point mutation occurs within the coding region of NPC1 (at D1005G). This mouse was discovered at the Jackson Laboratory as a result of chemically-induced mutagenesis. A noncomplementation test with other Npc1 alleles showed that NMF164 represents an allele of Npc1. The D1005G mutation is within the cysteine-rich luminal loop, where most common human mutations are found. In Npc1m/m mice, the npc1 mRNA levels appear relatively normal. Biochemical and histological analyses of liver, spleen, hippocampus, cortex, and cerebellum reveal abnormal cholesterol and glycolipid accumulation, glial activation, and progressive Purkinje cell loss. These mice exhibit characteristic gait and motor abnormalities, begin to lose weight at 8 weeks of age, and begin to die at 13 weeks of age; abnormalities very similar to the original NPC1−/− mouse. This model provides another useful tool for examining the pathogenesis of NPC disease as well as a model for testing potential new drug therapies.
Many different drug therapies have been attempted in order to treat NPC disease in efforts to either cure disease or to prolong the life of affected individuals. In mouse models for NPC disease, two drugs have shown promise. The first is Miglustat, also called NB-DNJ. Miglustat is an inhibitor of glycosphingolipid biosynthesis that reduces levels of all glucosylceramide-derived glycosphingolipids (GSLs) (Wraith and Imrie. 2009. Ther. Clin. Risk Manag. 5:877-887). Miglustat has shown efficacy in mouse models of GSL storage diseases, including Tay-Sachs and Sandhoff disease (Shapiro et al. 2009. Genet. Med. 11:425-433; Masciullo et al. 2010. J. Inherit. Metab. Dis. PMID: 20821051; Tallaksen and Berg. 2009. J. Inherit. Metab. Dis. PMID: 19898953). In cells affected by NPC disease, gangliosides, mostly GM2 and GM3, and neutral GSLs accumulate along with cholesterol within the late endosomes/lysosomes. When tested in the NPC1−/− mice, continuous administration of Miglustat starting at PND 7 significantly delayed the clinical onset and prolonged the lifespan by 49% (Davidson et al. 2009. PLoS One 4:e6951). In humans, a two-year study showed that Miglustat treatment stabilized NPC disease progression in 80% of patients (Patterson et al. 2009. J. Child Neurol. 25:300-305). Another drug that has shown some efficacy in animal models with NPC disease is hydroxypropyl beta-cyclodextrin (HPCD), a soluble cholesterol binder. HPCD can enter the cell interior and mobilize a portion of the cholesterol pool sequestered within the late endosomes/lysosomes in NPC cells. Single injection of HPCD to NPC1−/− mice at PND 7 prolonged their lifespan by 27% (Liu et al. 2009. J. Lipid Res. 51:933-944). Weekly treatment with HPCD was also shown to normalize cholesterol accumulation in NPC1−/− mice and to prolong survival (Ramirez et al. 2010. Pediatr. Res. 68:309-315). Problems associated with HPCD therapy include difficulty in crossing the blood brain barrier, and increases in macrophage activation in the lungs of treated NPC mice and NPC cats (Liu et al. 2009. J. Lipid Res. 51:933-944).
Other studies examining knockout of genes other than Acat1 gene that are involved in lipoprotein and cholesterol metabolism, such as Abca1, Apoa1, Apoe, Cyp46, Ldlr, Lxrβ, and SrbI, have not been associated with prolonged survival of NPC1−/− mice (Quan et al. 2003. Brain Res. Dev. Brain Res. 146:87-98; Repa et al. 2007. J. Neurosci. 27:14470-14480; Liu et al. 2008. J. Lipid Res. 49:663-669). Knockout of Siat9, a gene involved in biosynthesis of GM3 (Li et al. 2008. J. Lipid Res. 49:1816-1828), actually shortened the lifespan of NPC mice. Yet, over-expressing Rab9, a protein involved in membrane lipid trafficking, prolonged the lifespan of NPC1−/− mice by 22% (Kaptzan et al. 2009. Am. J. Pathol. 174:14-20).
As discussed above, acyl-CoA:Cholesterol Acyltransferase (ACAT) converts free cholesterol to cholesterol ester, and is a key enzymes in cellular cholesterol metabolism. While both ACAT1 and ACAT2 are present in the liver and intestine, the cells containing either enzyme within these tissues are distinct, suggesting that ACAT1 and ACAT2 have separate functions. As a result, both ACAT1 and ACAT2 are potential drug targets for treating diseases associated with cholesterol function, such as dyslipidemia and atherosclerosis. Dove et al. (2005. Arterioscler. Thromb. Vasc. Biol. 25:128-134) examined the role of ACAT1 deficiency on macrophage cholesterol efflux and cellular morphology. The authors were interested in understanding the mechanism for increased atherosclerosis in mouse models. They hypothesize that ACAT1−/− macrophages have a phenotype similar to NPC1−/− cells and that ACAT−/− increases intracellular vesicles that may release calcium stores and lead to apoptosis of the cells. Although inhibition of ACAT1 may be useful for treating a disease such as atherosclerosis, based on the findings of Dove et al. (2005. Arterioscler. Thromb. Vasc. Biol. 25:128-134), inhibition of ACAT1 in models of NPC disease would be expected to exacerbate the NPC phenotype and worsen disease.
It has now been found that contrary to predictions made in the art, inhibition of ACAT1 activity in NPC disease is an effective treatment that prolongs survival in animal models of NPC disease.